Novel supports carrying side chains, processes for obtaining these supports, process for attaching organic compounds having carbohydrate residues one said supports, products and reagents resulting from said chemical fixation

ABSTRACT

Process for chemically binding organic compounds containing carbohydrate residues, onto a support bearing at least one reactive --NH 2 , in which at least one --CH 2  OH group of the carbohydrate residue is transformed in a --CHO group, by oxidation and then the --CHO groups thus obtained are reacted with at least a reactive --NH 2  carried by the side chains covalently bound on a solid, insoluble support, the side chains are chosen from among amines, polyamines, diacids, amino-acids, hydrazines, and are eventually coupled, by the intermediary of their reactive --NH 2 , with a nitrogen-containing compound chosen from aliphatic or aromatic amines, aliphatic or aromatic hydrazines, or amino acids, comprising eventually jointly a --SH group and a --NH 2  group. 
     Products resulting from this process and biological reagents containing said products as their active constituents.

This is a continuation, of application Ser. No. 934,003, filed Aug. 16,1978, which is a division of application Ser. No. 740,840, filed Nov.11, 1976.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel insoluble solid supports carryingside chains bearing at least a reactive --NH₂, to processes forobtaining them, to processes for chemically binding organic compoundshaving carbohydrate residues on these supports, as well as to theproducts and to reagents resulting from said chemical fixation.

2. Description of the Prior Art

It is known that biologically active proteins (such as hormones,antigens and enzymes, for example) can be attached to insoluble solidsupports comprising latex spheres carrying lateral chains terminating inprimary amine functions, by reaction of active groups of the amino acidsof the protein chain with the primary amine groups of the support. Ithas also been proposed to use antigens fixed in the way which has justbeen very succinctly described above as biological reagents, but inassociation with developer reagents. The technique which has beenmentioned is described notably by R. S. MOLDAY, W. J. DREYER, A. REMBAUMand S. P. S. YEN in "The Journal of Cell Biology", Volume 64 (1975)75-88 and by R. W. LIM, R. S. MOLDAY, H. V. HUANG and SHIADO-PIN S. YENin "Biochimica et Biophysica Acta" 394 (1975) 377-387, which relates tothe fixation of antibodies on latex spheres by coupling the antibodiesthrough the intermediary of their primary amine functions via covalentbonds onto the latex spheres, which themselves have previously beenprovided with lateral chains terminating by a primary amine functionactivated by the action of activators such as glutaraldehyde, cyanogenbromide or watersoluble carbodiimide.

Such a process for coupling proteins through the intermediary of theiramino acids always presents a practical difficulty since to carry outthe coupling to biologically active amino acids of the protein chain hasthe effect of reducing, in certain cases, the biological activity of thefixed protein molecule. In addition, the known processes, notably thatof R. S. MOLDAY et al give rise to couplings of relatively low stabilityand of relatively low yields which reduce again their technical andeconomic interest.

OBJECTS AND SUMMARY OF THE INVENTION

It is consequently an object of the present invention to provide aprocess for chemically binding biologically active proteins on a supportcarrying side chains bearing at least a reactive --NH₂ without recourse,to effect the coupling, to biologically active groups of the amino acidsof the protein chain. In effect, proteins such as antibodies, antigensand a certain number of hormones and enzymes, in particular those thebinding of which on supports by chemical coupling has been described inthe prior art, are glycoproteins of which the prosthetic groups, namely,carbohydrate residues, do not contribute in any manner to the biologicalactivity of the molecule.

The applicant has now surprisingly developed a process for chemicallybinding organic compounds having carbohydrate residues, onto a supportthrough the intermediary of said residues, which in addition to notusing any active group of the bound organic compound, provide anattachment of very high stability, with satisfactory yields and withoutdiminishing, denaturing or lowering the specific activity of the boundorganic compound. The above references to the binding of an "organiccompound having carbohydrate residues" are not limited to the binding ofglycoproteins: in effect, the process of the invention permits thechemical binding of a given compound through the intermediary of itscarbohydrate residue to take place, and the process according to thepresent invention has a broader scope than the procedures hitherto knownin the prior art in that it is applicable not only to glycoproteins butalso to other biologically active organic compounds or to those ofindustrial interest, when they are coupled to a support such as thatdefined above, provided only that these organic compounds containcarbohydrate residues. Among such "organic compounds containingcarbohydrate residues" one can include, inter alia, not onlyglycoproteins but also polysaccharides and glycolipids.

DESCRIPTION OF THE INVENTION

According to the present invention there is provided a process forchemically binding organic compounds containing carbohydrate residuesonto a support bearing at least one reactive --NH₂, which processcomprises transforming, in a first step, at least one --CH₂ OH group ofthe carbohydrate residue of the organic compound into a --CHO group byoxidation, and, in a second step, reacting the so-obtained --CHO groupat least with a reactive --NH₂ carried by side chains attached to theinsoluble solid support so as to chemically bind the organic compound onsaid support, and in cases where the reactive --NH₂ is a primary aminegroup, the resulting Schiff base is stabilized, by chemical reduction.

According to one preferred manner of carrying out the process of theinvention, the oxidation of the --CH₂ OH groups to --CHO groups iseffected using sodium periodate.

According to another preferred manner of carrying out the process of theinvention, the oxidation of the --CH₂ OH groups to --CHO groups iscarried out enzymically.

According to another preferred manner of carrying out the process of theinvention, an organic compound containing an appropriate carbohydrateresidue, prior to the transformation of at least one of the --CH₂ OHgroups to a --CHO group by oxidation, is subjected to a treatment withneuraminidase, when the terminal residue of the carbohydrate chain is asialic acid, which treatment eliminates said sialic acid.

Advantageously, where the step of oxidising the --CH₂ OH group to a--CHO group is effected enzymically, the enzyme used is selectedaccording to the nature of the last-but-one residue of the carbohydratechain, from the group comprising the oxidases which oxidisecarbohydrates, namely, glucose oxidase, fucose oxidase and galactoseoxidase i.e. according to whether the last-but-one residue of thecarbohydrate chain is respectively glucose, fucose or galactose.

According to the invention, when the side chain of said support carriesa terminal primary amine group, the reaction between the --CHO group ofthe organic compound to be bound to the support and the said --NH₂ groupgives rises to a Schiff base, which is stabilised during a third step ofthe process of the invention, by chemical reduction, using preferablyNaBH₄.

Such stabilisation is not necessary in the case where the side chainsare terminated by a --NH--NH₂ group, which forms a stable hydrazone inthe presence of --CHO groups; in cases where the side chain carried bothas --NH₂ and a --SH, their interaction with a --CHO group also givesrise to the formation of a stable heterocyclic compound, which, forinstance, can be a thiazolidine in the case of cystein, where the --SHis in a β-position with respect to the --NH₂.

The process according to the present invention for chemically bindingcompounds containing carbohydrate residues onto solid, insolublesupports having side chains with at least a reactive --NH₂, can giverise to coupled products which are useful, in particular as biologicalreagents.

The applicant has established that, surprisingly, the reactivity andstability of the coupled products so obtained are greatly increased ifthe length of the side chain is itself sufficiently increased. It hasalso been established that, surprisingly, diagnostic reagents comprisingthe aforementioned coupled products possess qualities of sensitivity andprecision which are not possessed by diagnostic reagents of similar typeproposed in the prior art.

The supports used for the chemical binding of the organic compoundshaving at least one --CHO group, are insoluble, solid supports selectednotably from the group which comprises latex spheres, agarose ordextrane beads, activated glass beads or the like, on which arecovalently bound side-chains bearing at least a reactive --NH₂ resultingfrom the binding onto the said supports, of compounds selected among theamines, polyamines, diacids, amino-acids, aliphatic or aromatichydrazines bearing eventually an acid group.

Among these supports, those which are new and are within the scope ofthe present invention, are supports on which are covalently boundside-chains bearing at least a reactive --NH₂ resulting from the bindingon said supports, of a compound selected among the aromatic amines,polyamines other than diamines, substituted amino-acids, aliphatic oraromatic hydrazines bearing eventually an acid group.

According to a particularly advantageous embodiment of the invention,the side-chains bound to the supports, be they known supports or the newones according to the present invention, are coupled through theintermediary of their reactive --NH₂, with a nitrogeneous compound alsocontaining at least a reactive --NH₂ and chosen notably among thealiphatic or aromatic amines and/or aliphatic or aromatic hydrazines, oramino-acids, notably those containing both an --NH₂ and a --SH capableof forming an heterocycle in the presence of the --CHO group of theorganic compound to be bound.

Advantageously, the insoluble solid supports on which are bound theaforementioned side chains carry carboxyl groups through which the sidechains are bound to said supports.

In another preferred embodiment of supports according to the invention,the insoluble solid supports carry --NH₂ groups through which theabovementioned side chains are bound to the support.

According to the invention, the amines or polyamines bound to the solidinsoluble supports are selected from aromatic diamines, polyamines otherthan aliphatic diamines, and substituted amino-acids.

Advantageously, there are bound to the abovementioned supports: aromaticdiamines, polyamines containing primary and secondary amine groupshaving, for example, the following formula:

    NH.sub.2 (CH.sub.2).sub.x NH(CH.sub.2).sub.y NH.sub.2

where x≧3 and y≧3

and having the formula:

    NH.sub.2 (CH.sub.2).sub.x NH(CH.sub.2).sub.y NH(CH.sub.2).sub.z NH.sub.2

where x≧3, y≧4 and z≧3,

or appropriate substituted amino-acids, among which can be includedcompounds containing a --SH group as in cystein of the formula: ##STR1##

According to an advantageous embodiment of the invention, the sidechains bound to the support result from coupling an aliphatic oraromatic amine, such as a diamine, polyamine or amino-acid, with otheraliphatic or aromatic amines such as, in particular: diamines,polyamines, aliphatic or aromatic hydrazines or their derivatives.

According to a particularly advantageous embodiment of the invention,the solid insoluble support carries hexamethylenediamine (HMD) chainsadvantageously coupled through the intermediary of their reactive --NH₂,to adipic dihydrazide, to p-hydrazino-benzoic acid, or the like.

According to a further particularly advantageous embodiment of theinvention, the insoluble solid support carries polyamine chains, such asspermidine, diaminopropylamine and spermine, for example, advantageouslycoupled through the intermediary of their reactive --NH₂, to adipicdihydrazide, to p-hydrazinobenzoic acid or the like.

According to a further advantageous embodiment of the present invention,the insoluble solid supports carry side chains comprising an amino-acidsuch as β-alanine or ε-aminocaproic acid, for example, advantageouslycoupled to diaminopropane, to adipic dihydrazide, to p-hydrazino-benzoicacid or to analogous compounds.

According to another advantageous embodiment of the invention, theinsoluble solid supports carry side chains ending with an amino-acidcarrying a --SH group, as for example cysteine.

According to an advantageous provision of said embodiment, theamino-acid carrying a --SH group is coupled to an aliphatic or aromaticdiamine.

The present invention also provides a process for binding side chainshaving a reactive --NH₂ onto an insoluble solid support selected notablyfrom latex spheres, dextrane or agarose beads, beads of activated glassand the like, which comprises binding the aforementioned side chainsonto the support in the presence of an appropriate condensation agent,the fixation process including eventually a subsequent coupling step ofsaid chains with nitrogen-containing compounds advantageously selected,in particular, among amines, aliphatic or aromatic hydrazines,amino-acids, which coupling step is also advantageously effected in thepresence of an appropriate coupling agent, such as carbodiimide orN-hydroxy-succinimide.

According to the invention, one advantageously uses as the condensationand/or coupling agent glutaraldehyde or a totally or partiallywater-soluble carbodiimide of the general formula:

    R--N═C═N--R

in which R represents notably an alkyl radical having 2 to 12 carbonatoms, and particularly an ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, tert-butyl, isobutyl, amyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl; a cycloalkyl radical having 5 or 6 carbonatoms; a mono-aryl substituted lower alkyl radical such as, for example,a benzyl or α- or β-phenylethyl radical; a monoaryl radical such as, forexample, a phenyl, morpholino- or piperidyl radical; a lower alkoylradical substituted by a morpholinyl group, such as, for example, anethylmorpholinyl radical; a lower alkyl radical substituted by apiperidyl group, such as, for example, an ethylpiperidyl radical; alower dialkylamino radical; a lower alkyl radical substituted by apyridyl group, such as, for example, an α-, β-, or γ-methyl- orethyl-pyridyl radical; their acid addition salts with acids and theirquaternary ammonium salts, the two R radicals being identical ordifferent.

According to a preferred embodiment of the process of the invention, thecovalent binding of the side chains on the support is effected in anon-agglutinant buffer, containing no free --NH₂ groups and at a pHbetween 6,0 and 8,8.

According to another preferred embodiment of the process of theinvention, the covalent binding is followed by dialysis against the samebuffer as that used for the binding.

The solid insoluble supports having side chains obtained according tothe present invention are themselves novel products, which are capableof numerous industrial applications.

One of these applications consists in using them as supports for organiccompounds having at least one --CHO group, chemically bound on saidsupports by reaction between their --CHO group and the reactive --NH₂ ofthe side chains of said supports.

This application presents a particular interest in the case of thebinding of organic compounds comprising carbohydrate residues, which iseffected through the intermediary of said carbohydrate residues, inmodifying initially by oxidising two adjacent functional groups carriedby the latter into --CHO groups, then bringing these --CHO functions toreact at least with a reactive --NH₂ of the side chains to the solidinsoluble support.

The oxidation of the carbohydrate residues of the organic compound to bebound to the support is preferably effected at about pH 6.

Advantageously, the excess oxidising agent is eliminated at the end ofthe abovementioned oxidation reaction, by all appropriate means, such asdialysis or addition of an agent capable of being oxidized by theoxidizing agent, such as sodium sulfite, glycerol, glucose and the like.

The interest of the above application is increased when the organiccompounds having carbohydrate residues at least partially oxidised to a--CHO, bound to the supports according to the present invention, areselected from glycoproteins, polysaccharides and glycolipids; in effect,antigens, antibodies a certain number of enzymes and hormones belongingto the group of glycoproteins and their fixation to an insoluble solidsupport through the intermediary of their carbohydrate residues whichhave previously been oxidised to an aldehyde, allows their free proteinmoiety or their free lipidic moiety (for glycolipids) to be utilised incarrying out tests allowing the precise detection, with highsensitivity, which can be of the order of nanograms of detectableantigen, using the insoluble diagnostic agents realised in this way.

According to the present invention, one again improves the ease of usingbiological reagents which make them also form further aspects of thepresent invention, by chemically binding glycoproteins, glycolipids orpolysaccharides with solid insoluble supports carrying side chainsbearing a reactive --NH₂, selected from basic dyes.

One thus obtains coloured biological reagents which allow immunologicalreactions to be revealed (with antigens or their antibodies inparticular) by direct reaction, notably agglutination reactions withouthaving hereafter to resort to the intermediary of red corpuscles, thereading of the results of agglutination reactions in thus found to begreatly facilitated.

According to the invention, these solid, insoluble coloured supports areprepared by binding side chains on the supports in an appropriatebuffer, in the presence of a coupling and/or condensation agent added intwo successive steps, and selected from, notably, glutarladehyde,N-hydroxy-succinimide and totally or partially water-solublecarbodiimides, the binding process being followed by desorption ofremaining non-coupled reagents.

According to an advantageous method of preparing the coloured supports,these are maintained, before use, in a nonagglutinating buffer.

Numerous biological reagents can be prepared and put to use inaccordance with the present invention, these ragents distinguishingthemselves by their excellent stability and their high sensitivity.

The coupled products resulting from the chemical binding of organiccompounds having carbohydrate residues on a solid insoluble supporthaving side chains bearing at least a reactive --NH₂ according to thepresent invention, can also be used as catalysts in numerous chemical,biochemical and biological reactions.

Having regard to the foregoing, the invention includes many alternativeembodiments which will be apparent from the following description.

The invention has for its aim more particularly the processes ofchemical binding of organic compounds containing carbohydrate residues,on a support having at least one reactive --NH₂, according to theforegoing description, to the products thereof, to reagents andcatalysts comprising the coupled products obtained in putting theseprocesses into operation and the means themselves of putting intooperation these processes and obtaining the said products, reagents andcatalysts.

The invention will be better understood with the aid of thesupplementary description which follows, which refers to examples ofsupports and novel products according to the invention, as well as toexamples of the preparation of diagnostic reagents according to thepresent invention, of which the high sensitivity and precision are shownin the frame-work of tests by radioactivity and nephelometry.

It must be well understood, however, that these examples are givensolely for the purpose of illustrating the objects of the invention, ofwhich they do not constitute in any manner a limitation.

EXAMPLES I.--EXAMPLES OF PREPARATION OF SOLID INSOLUBLE SUPPORTSACCORDING TO THE PRESENT INVENTION A. EXAMPLES OF PREPARATION OF SOLIDINSOLUBLE SUPPORTS CARRYING SIDE CHAINS ABLE TO BE COUPLED TO ANOTHERNITROGEN-CONTAINING COMPOUND CARRYING A REACTIVE --NH₂ A₁ --PREPARATIONOF SUPPORTS FROM CALIBRATED SPHERICAL PARTICLES OF CARBOXYLATEDPOLYSTYRENE BEARING SIDE CHAINS CARRYING AT LEAST A REACTIVE --NH₂EXAMPLE 1 PREPARATION OF A SUPPORT CARRYING HEXAMETHYLENEDIAMINE SIDECHAINS

To 12 mg of "ESTAPOR PSI₆₈ " (Rhone-Progil Registered Trade Markdesignating calibrated spherical particals of carboxylated polystyreneof diameter of the order of 0.22μ) bearing free --COOH groups insuspension in a borate buffer of composition 0.14 M NaCl, 0.01 MBorate-HCl of pH 8.1 (BBS) were added 80 μmoles of hexamethylene-diamine(HMD). The mixture was shaken at 20° C. for three hours in the presenceof 0.02 M of 1-ethyl 3-(3-dimethylaminopropyl-carbodiimide)chlorhydrate(EDC). Thereafter, the suspension was dialysed against BBS buffer untilits O.D. at 230 nm was equal to zero.

One obtained a support of "Estapor" carboxylated hexamethylenediamine("Estapor"-HMD).

EXAMPLE 2 PREPARATION OF A SUPPORT BEARING SIDE CHAINS CONSTITUTED BYPOLYAMINE

The procedure described in Example 1 was followed, except that sperminewas used in place of HMD.

EXAMPLE 3 FIXATION OF A SIDE CHAIN CONSTITUTED BY AN AMINO ACID ON ANINSOLUBLE SUPPORT

To 20 mg of "Estapor PSI₄₂ " carrying free --COOH groups in suspensionin a BBS buffer of pH 8.5 were added 20 μmoles of ε-amino-caproic acidof which the primary amino group is not in the alpha position. Themixture was shaken for three hours at about 20° C. in the presence of0.02 M of EDC. Thereafter, the suspension was dialysed against BBSbuffer until the O.D. at 230 nm was equal to zero. The fixation of theamino acid on the support was effected through the intermediary of theCONH(CH₂)₅ COOH groups which formed at the level of the free --COOHgroups of the support.

EXAMPLE 4 FIXATION OF CRESYL VIOLET

To 10 mg of "Estapor" (registered trade mark of Rhone-Progil todesignate carboxylated polystyrene) having free --COOH groups insuspension in 5 ml of 0.14 M NaCl-0.01 M Borate-HCl of pH 8.1 (BBS), isadded a solution of Cresyl violet obtained by filtration on a 0.22μ"Millipore" of a suspension of 20 mg of ultrasonically-treated Cresylviolet. The mixture was shaken for 12 hours at 20° C. in the presence of0.05 M of 1 ethyl-3(3-dimethylaminopropylcarbodiimide)hydrochloride(EDC). At the end of this period, one renewed the addition of EDC andcontinued the shaking for 12 hours.

Thereafter, the suspension was centrifuged at 20,000 g for 1 hour andthe residue taken up in 0.1 M acetone buffer of pH 5.5 and recentrifugedto eliminate the excess Cresyl violet. This operation was repeated untilthe supernatant was clear. One then eliminated the acetate by forming ahydrochloride by three successive washings with 0.1 N HCl, followed bytwo washings with 0.1 N NaOH, in order to regenerate the aminefunctions, then two washings in BBS of pH 8.8 to eliminate the excessNaOH.

The obtained product was maintained in a 0.1 M phosphate buffer of pH 7.

EXAMPLE 5 FIXATION OF BASIC FUCHSINE

One proceeded in the manner described in EXAMPLE 4, but in place of the20 mg of Cresyl violet, one added 2 ml of a saturated solution of basicfuchsine.

EXAMPLE 6 PREPARATION OF SUPPORTS FROM DOW 816 CARBOXYLATED POLYSTYRENE

The coupling reactions described above on "Estapor" carboxylatedpolystyrenes obtained from Rhone-Poulenc and on glass beads have equallybeen carried out on Dow 816 carboxylated polystyrene spheres obtainedfrom Dow Chemical Corporation, in proceeding to adapt the method ofcarrying out the coupling reactions of the side chains on theaforementioned spheres, one takes account of a number of available--COOH groups on the spheres with regard to those on the "Estapor"spheres.

To 180 mg of Dow 816 particles were added 560 mg of hexamethylenediamine(HMD) in 20 ml (final) of 0.14 M NaCl, 0.01 M borate-HCL of pH 8.1. Some1-ethyl3-3-dimethylaminopropyl)carbodiimide (EDC) was added to a finalconcentration of 0.5 M in two successive additions. After shaking at 4°C. for 8 hours, the Dow spheres were washed by three successivecentrifugations at 20,000 g for 30 minutes with the same buffer andfinally suspended in 13 ml of 0.1 M phosphate buffer of pH 6.

A₂ --PREPARATION OF SUPPORTS FROM GLASS BEADS BEARING SIDE CHAINSCARRYING A REACTIVE --NH₂ EXAMPLE 7 FIXATION OF HMD SIDE CHAINS ON GLASSBEADS

To 1 g of glass beads bearing free --COOH groups (sold under thecommercial designation "CPG/carboxyl" by Corning Glass Works (dimensionof pores: 550 A; diameter: 177-840 microns), were added 5 ml of 0.1 Mphosphate buffer of pH 7.0 and the suspension was degassed under vacuum.50 μmoles of HMD were added in solution in the same buffer and themixture was shaken gently at 4° C. for 18 hours in the presence of 0.5 Mof EDC. Thereafter, the glass beads were washed with phosphate buffer.

The terminal --NH₂ groups of the HMD side chains fixed to the glassbeads were coupled to a hydrazine in the manner described in EXAMPLE 13in the case of an aliphatic hydrazine or in the manner described inEXAMPLE 11 in the case of an aromatic hydrazine.

EXAMPLE 8

BINDING OF A POLYAMINE ON A SIDE CHAIN TERMINATED BY ANN-HYDROYL-SUCCINIMIDE GROUP CARRIED BY GLASS BEADS

2 g of commercially available glass beads known under the designation"CPG/N-OH succinimide" (sold by Corning Glass Works) and represented bythe formula: ##STR2## were suspended in 10 ml of BBS buffer at pH 8.2and degassed under vacuum and 40μ moles of spermine added thereto. Themixture was shaken gently for 16 hours at 4° C., then washed with BBSbuffer to eliminate the reaction products.

The --NH₂ terminals of polyamine residues were themselves coupled toaliphatic or aromatic hydrazines according to the technique described inEXAMPLES 11 and 13.

A₃ --PREPARATION OF SUPPORTS FROM AGAROSE GEL BEADS BEARING SIDE CHAINSCARRYING A REACTIVE --NH₂ EXAMPLE 9 BEADS OF AGAROSE GEL CARRYING SIDECHAINS OF ε-AMINO-CAPROIC ACID

"CH-SEPHAROSE" (registered trade mark of Pharmacia, Upsala, Sweden todesignate beads of agarose gel) already carry side chains ofε-amino-caproic acid.

B--EXAMPLES OF PREPARATION OF SOLID INSOLUBLE SUPPORTS CARRYING SIDECHAINS ABLE TO REACT WITH THE --CHO GROUPS OF THE COMPOUNDS TO BE BOUNDONTO SAID SUPPORTS B₁ --BINDING OF TERMINAL FUNCTIONAL GROUPSCONSTITUTED BY HYDRAZINES B₁.1 --CONSTITUTED BY AROMATIC HYDRAZINESEXAMPLE 10 PREPARATION OF AN "ESTAPOR"-HMD-P-HYDRAZINOBENZOATE SUPPORT

4 mg of "Estapor"-HMD of 0.22μ diameter were suspended in 2 ml of boratebuffer comprising 0.14 M NaCl, 0.01 M borate-HCl, of pH 8.8. To thissuspension were added 22μ moles of hydrazinobenzoic acid dissolved inBBS. The volume was adjusted to 4 ml with BBS. After the addition of 10mg of 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide, the tube wasshaken at ambient temperature (20° C.) for 2 hours. Its contents werethen transferred into a dialysis sack and dialysed at ambienttemperature for 24 hours against 300 ml of BBS (three changes). One thusobtained a "Estapor"-HMD-p-hydrazino-benzoate support.

EXAMPLE 11 FIXATION OF A SIDE CHAIN CONSTITUTED BY A POLYAMINE COUPLEDTO AN AROMATIC HYDRAZINE ONTO AN INSOLUBLE SUPPORT

To 7 mg of "Estapor"-spermine prepared as described in Example 2 aboveand having the formula: ##STR3## in suspension in 15 ml of BBS buffer ofpH 8.1 were added 100μ moles of p-hydrazino-benzoic acid in aqueoussolution. The mixture was shaken for 24 hours at ambient temperature(about 20° C.) in the presence of 0.05 M of EDC. Thereafter, thesuspension was dialysed against BBS buffer of pH 8.8 until the O.D. at230 nm was equal to zero.

EXAMPLE 12 FIXATION OF AROMATIC HYDRAZINE

To 10 mg of "Estapor" in the --NH₂ form (aromatic amine) in suspensionin 10 ml of 0.1 M phosphate buffer of pH 7, is added 50μ moles ofp-hydrazinobenzoic acid in solution in a 0.1 M borate buffer of pH 8.The mixture was shaken at 20° C. for 12 hours in the presence of 0.05 Mof EDC. At the end of this time, one renewed the addition of EDC andcontinued the shaking for 12 hours. At the end of this second period,the suspension was centrifuged at 20,000 g for 1 hour and the residuetaken up in 0.1 M borate buffer of pH 8.1 and recentrifuged to eliminatethe p-hydrazinobenzoic acid which had not been coupled. This washing wasfollowed by two other washings (0.01 M borate buffer of pH 8.1) then twowashings with 0.1 M phosphate of pH 7. The obtained product wasmaintained in the last-mentioned buffer.

B₁.2 --CONSTITUTED BY ALIPHATIC HYDRAZINES EXAMPLE 13 FIXATION OF A SIDECHAIN COMPRISED BY AN ALIPHATIC DIAMINE COUPLED TO AN ALIPHATICHYDRAZINE ON AN INSOLUBLE SUPPORT

6.0 mg of "Estapor"-HMD prepared as described in Example 1 above andhaving the formula: ##STR4## suspended in 0.1 M of phosphate buffer ofpH 7 were shaken at 20° C. for 1 hour in the presence of glutaraldehydeat a final concentration of 1.25%. Thereafter, this suspension wasdialysed against 0.1 M of phosphate buffer of pH 7.0 for 18 hours. Tothe suspension obtained after dialysis, were added 20μ moles of adipicdihydrazide in aqueous solution. The mixture was shaken for 16 hours at20° C. and then dialysed to eliminate the excess of adipic dihydrazide.

B₂ --BINDING OF TERMINAL FUNCTIONAL GROUPS CONSTITUTED BY A PRIMARYAMINE FUNCTION

EXAMPLE 14

BINDING OF A SIDE CHAIN CONSTITUTED BY AN AMINO ACID COUPLED TO ADIAMINE ON A SOLID INSOLUBLE SUPPORT

Diaminopropane is bound to the "Estapor" beads which carryε-amino-caproic acid according to EXAMPLE 3, by the following procedure:

The contents of the dialysis bag were transferred to a tube containing25μ moles of diaminopropane. The mixture was shaken for three hours at20° C. in the presence of 0.05 M of EDC and then dialysed against a BBSbuffer of composition 0.14 M NaCl, 0.01 M Borate-HCl, pH 8.8, the end ofwhich the diaminopropane was coupled to the ε-amino-caproic acid fixedon the insoluble support.

EXAMPLE 15 COUPLING OF HMD ON BEADS OF AGAROSE GEL CARRYING SIDE CHAINSOF ε-AMINO-CAPROIC ACID

The coupling of HMD onto the substituted "SEPHAROSE" beads of EXAMPLE 9,is effected according to the procedure described in EXAMPLE 7.

The --NH₂ terminals of the HMD substituent of the ε-amino-caproic acidside chains were themselves coupled with an aliphatic or aromatichydrazine which is substituted on the said --NH₂ terminal groups inaccordance with two methods described respectively in EXAMPLE 11 and 13.

B₃ --BINDING ON A SOLID INSOLUBLE SUPPORT, OF SIDE CHAINS CARRYINGTERMINAL FUNCTIONAL GROUPS COMPRISING SH--CH₂ --CH--NH₂ GROUPS ABLE TOFORM A THIAZOLIDINE DERIVATIVE IN THE PRESENCE OF THE --CHO GROUP OF THECOMPOUND TO BE BOUND ON A SAID SUPPORT EXAMPLE 16 FIXATION ON ANINSOLUBLE SUPPORT HAVING FREE --NH₂ GROUPS OF AN AMINO ACID HAVING --SHGROUPS

To 2 mg of "Estapor"-NH₂ in suspension in phosphate buffer of pH 6.5were added 6μ moles of cysteine hydrochloride of formula HSCH₂ CH NH₂COOH, HCl dissolved in water. The mixture was shaken at 20° C. for 16hours in the presence of 0.05 M of EDC. Thereafter, the "Estapor"spheres on which the cysteine was fixed were washed on a 0.22μ Milliporefilter to eliminate the excess cysteine and the reaction products. Thespheres which had been modified in that way were again put in suspensionin 2 ml of phosphate buffer of pH 6.5.

EXAMPLE 17 FIXATION OF CYSTEINE ON DOW 816 CARBOXYLATED POLYSTYRENE-HMD

To the suspension of Dow 816 carboxylated polystyrene-HMD obtained inEXAMPLE 6, are added 18 mg of cysteine-HCl dissolved in 2 ml of 0.1 Mphosphate buffer of pH 6 and then EDC to a final concentration of 0.05M. After 18 hours at 4° C., the spheres were washed as described inEXAMPLE 6 above.

EXAMPLE 18 EXAMPLES OF OXIDATION OF THE COMPOUNDS TO BE BOUND ONTO THESUPPORTS

The determinating role of the oxidation according to the presentinvention, in the fixation of the oxidised compounds on the solidinsoluble supports, is demonstrated in the following EXAMPLE 18.

EXAMPLE 18 DEMONSTRATION OF THE DETERMINATING ROLE OF FREE --CHO GROUPSOF GLOBULINS IN THE FIXATION OF THE LATTER ON AN INSOLUBLE SUPPORT

Gammaglobulins were oxidised as described but prereduced in the presenceof NaB³ H₄ so that they are tritium-labelled and do not have free --CHOgroups. Aliquots of these gammaglobulins (0.3 ml) containing 8400 cpmwere put in contact with the samples set forth in the Table below in thepresence of 0.1 M phosphate buffer of pH 6.0. After 16 hours at 4° C.,the samples were filtered on a 0.22μ Millipore filter and washed fourtimes with 5 ml of the following buffer:

0.1% Bovine Serum Albumin

0.1% Triton ×100

0.14 M NaCl, 0.01 M Borate-HCl pH 8.8

    ______________________________________                                                                         cpm retained on                              Sample         Quantity Volume   filter                                       ______________________________________                                        (A)  Buffer        --       2      319                                        (B)  Estapor 68    500 μg                                                                              2      311                                        (C)  Estapor NH.sub.2                                                                            500 μg                                                                              2      276                                        (D)  Estapor NH--NH.sub.2                                                                        500 μg                                                                              2      204                                        ______________________________________                                    

It appears from the results gathered in the foregoing Table that: (1) acertain quantity of radioactivity is retained on the filter; (2) thewashing with the abovesaid buffer diminishes considerably the adsorptionon the filters themselves. In the latter case, the adsorption on thefilters due to the gammaglobulins+buffer alone represents approximately20% of the total radioactivity of the aliquot (1:80 cpm) which remainson the filter; (3) the quantity of radioactivity retained on the filteris not increased significantly in the presence of "Estapor 68-COOH", of"Estapor 68-NH₂ " or of "Estapor-NH-NH₂ ". In fact, the values are equalor less than the results obtained with the buffer alone (319 cpm). Thus,there is no adsorption of the gammaglobulins on the three types of"Estapor" used. Moreover, in order to make this filtration proceduresignificant, the radioactivity coupled to the insoluble supports in thepresence of the oxidised gammaglobulins must exceed the 20% found withthe nonoxidised gammaglobulins.

COMPARISON BETWEEN THE RADIOACTIVITY CONTENT ASSOCIATED WITH THE"ESTAPOR-NH₂ ", AND THE OXIDISED AND NON-OXIDISED GAMMAGLOBULINS AFTERREDUCTION WITH NaB³ H₄

    ______________________________________                                        Reagents brought                                                                          Volumes                                                           together    ml         Oxidised Non-oxidised                                  ______________________________________                                        ESTAPOR-NH.sub.2 in                                                                       2.00       1 mg     1 mg                                          0.1M PO.sub.4, pH 6.0                                                         Oxidised horse                                                                            0.10       2 mg     --                                            gammaglobulins                                                                Non-oxidised horse                                                                        0.10       --       2 mg                                          gammaglobulins                                                                0.1M Borate, pH 8.8                                                                       0.90       +        +                                             NaB.sup.3 H.sub.4 in H.sub.2 O                                                            0.20       2 μCi 2 μCi                                      ______________________________________                                    

After contacting during 18 hours at 4° C., the content of each bag isdialysed during 5 days against BBS pH 8.8 with three or four changes perday, until the dialysis liquid contains no more radioactivity. Analiquot of each preparation is then filtered on Millipore 0.22μ andwashed with the buffer 0.1% SAB, 0.1% Triton×100, BBS pH 8.8.

The optical density at 550 nm is measured on another aliquot in order tofind the "Estapor" concentration.

    ______________________________________                                        Sample              cpm/mg                                                    ______________________________________                                        Estapor 68-NH.sub.2 --oxidised                                                                    62 466                                                    gammaglobulin                                                                 Estapor 68-NH.sub.2 non-oxidised                                                                  11 135                                                    gammaglobulin                                                                 ______________________________________                                    

The radioactivity found with Estapor 68-NH₂ in the presence ofnon-oxidised gammaglobulins represents only 17.8% of the radioactivityfound in association with the Estapor 68-NH₂ in the presence of oxidisedgammaglobulins. (This value is of the same magnitude as the value due tothe adsorption of gammaglobulins on filters in the absence of Estapor).

On the other hand, with the oxidised gammaglobulins the quantity orradioactivity associated with Estapor-NH₂ increases five times.

Some examples of oxidation of various compounds containing carbohydrateresidues, by oxidation of at least one of their --CH₂ OH groups in --CHOgroups, will be given hereafter.

A. OXIDATION OF GAMMAGLOBULINS EXAMPLE 19 CHEMICAL OXIDATION OFGAMMAGLOBULINS IN SOLUTION

Gammaglobulins obtained from the goat, the rabbit and the horse werepurified by NH₄)₂ SO₄ precipitation followed by chromatography onDEAE-cellulose.

To 60 mg of horse immunogammaglobulins (containing antipolyamineantibodies) in solution in 2 ml of 0.1 M phosphate buffer of pH 6 wereadded 0.2 ml of 0.15 M NaIO₄ soluble in water.

The mixture was left at 4° C. and protected from light for 3 hours, thendialysed against a 0.14 M NaCl-0.1 M phosphate buffer of pH 6 for 1hour, with three changes. At this pH, th risk of formation of Schiffbridges between the reactive aldehyde groups obtained by oxidation byNaIO₄ and the --NH₂ groups, be it those of lysine or of terminal aminogroups of proteins is reduced.

EXAMPLE 20 CHEMICAL OXIDATION OF ANTIGAMMAGLOBULINS-ANTIBODIES

To 520 μg of goat anti-rabbit gammaglobulins in 0.1 ml of BBS whereadded 0.1 ml of 0.03 M NaIO₄. The mixture was left at 4° C., protectedfrom light, for 3 hours and then dialysed against BBS for 2 hours withthree changes of buffer.

EXAMPLE 21 CHEMICAL OXIDATION OF GAMMAGLOBULINS

To 60 mg of horse immunoglobulins in solution in 2 ml of 0.1 M phosphatebuffer of pH 6 were added 0.2 ml of 0.15 M NaIO₄ soluble in water.

The mixture was left at 4° C. protected from the light for three hoursat the end of which the action of the NaIO₄ was arrested by addition ofglycerol to a final concentration of 0.15 M. After 30 minutes at 4° C.,the reaction products were dialysed at 18 hours at 4° C. with severalchanges against 0.1 M phosphate buffer of pH 6.

B. OXIDATIONS OF VIRUSES EXAMPLE 22 PREPARATION OF OXIDISED MEASLESVIRUS

To 5 ml of an antigenic preparation of measles virus containing 16 mg ofprotein were added 0.5 ml of 0.15 M NaIO₄ in a 0.1 M sodium phosphatebuffer of pH 6.

This mixture was left at 4° C. and protected from the light for 3 hours.

0.5 ml of 0.15 M Na₂ SO₃ was then added to arrest the action of theNaIO₄.

After 30 minutes at 4° C., the mixture was dialyzed for 18 hours at 4°C. in order to eliminate the excess oxidation products.

EXAMPLE 23 PREPARATION OF OXIDISED CANINE DISTEMPER VIRUS

The procedure carried out for oxidising the Canine Distemper virus isthat which is described in the foregoing Example 22.

EXAMPLE 24 PREPARATION OF OXIDISED INFLUENZA VIRUS

The procedure carried out for oxidising the influenza virus is thatwhich is described in the foregoing Example 22.

EXAMPLE 25 PREPARATION OF OXIDISED GERMAN MEASLES VIRUS

The procedure carried out for oxidising the German Measles virus is thatwhich is described in the foregoing Example 22.

C/OXIDATION OF HORMONES EXAMPLE 26 PREPARATION OF OXIDISED HUMANCHORIONIC GONADOTROPHIN (HCG)

The procedure carried out for oxidising this hormone is that describedin the foregoing Example 19 and 20.

III.--EXAMPLES OF COUPLING OF COMPOUNDS CONTAINING CARBOHYDRATE RESIDUESAT LEAST ONE --CH₂ OH GROUP OF WHICH HAS BEEN OXIDISED TO A --CHO GROUP,ON THE SUPPORTS OF THE INVENTION EXAMPLE 27 EXAMPLE OF COUPLING EFFECTEDON A TERMINAL --NH₂ : PREPARATION OF "ESTAPOR"-HMD-ANTIPOLYAMINEANTIBODY REAGENT BY FIXING ANTIBODIES ON THE SUPPORT

To prepare a reagent according to the invention using antipolyamineantibodies, which are utilised elsewhere for research and for estimatingpolyamines in the blood of patients, one fixes these antibodies bysubstrate affinity chromatography on glass supports containing sperminethen one oxidises them with an 0.06 M solution of NaIO₄. After 30minutes at 4° C. in darkness, the antibodies are washed in BBS buffer ofpH 8.8 in order to eliminate excess NaIO₄.

2 ml of "Estapor"-HMD (10 mg) (prepared as indicated in Example 1 above)were mixed with the oxidised antibody. The suspension was shaken gentlyby a rotary movement at 4° C. for 14 hours. The Schiff-bases so formedwere stabilised by treatment for 5 hours at 4° C. with NaBH₄ (3μ moles)under intermittent agitation. The supernatant was recovered, the residuewas rewashed four times with BBS and all the supernatants were reunited.

EXAMPLE 28 EXAMPLE OF COUPLINGS EFFECTED ON A HYDRAZINE: PREPARATION OFREACTIVE "ESTAPOR"-HMD-P-HYDRAZINOBENZOATE-ANTI IgG ANTIBODIES

"Estapor"-rabbit anti IgG antibodies, which constitute a utilisationmodel using antibodies in solution, were prepared. The final preparationpresented a very large spectrum of utilisation because it allowed allimmunological reactions in which rabbit IgG are used to be detected bythe indirect method.

0.1 ml of the solution obtained in Example 20 (260 μg) where then addedto 1.7 mg of "Estapor"-HMD-p-hydrazinebenzoate. The mixture was shakenfor 3 hours at 4° C.

As in Example 27 above, after the described treatment, the beads of"Estapor" containing the antibodies were washed and concentrated bysedimentation on a layer of 60% sucrose through a solution of 10%sucrose (100 000 g for 1 hour at 4° C.). The "Estapor" concentrated atthe interface and was dialysed to eliminate the excess sucrose.

EXAMPLE 29 EXAMPLE OF COUPLING EFFECTED ON A TERMINAL --SH: COUPLING OFAN INSOLUBLE SUPPORT HAVING FREE TERMINAL --SH GROUPS TO --CHO GROUPS OFOXIDISED GAMMAGLOBULINS

To 2 mg of "Estapor-SH" were added 1 mg of oxidised gammaglobulins asdescribed in Example 10. After 30 minutes of contact at 4° C., oneobserved a floculation of the "Estapor-SH" particules by formation ofthiazolidines. This floculation is not produced with "Estapor-NH₂ " norwith "Estapor--SH" on which one has fixed non-oxidised gammaglobulines.

IV.--EXAMPLES OF ISOTOPIC LABELLING OF THE PRODUCTS OF THE INVENTIONEXAMPLE 30 PREPARATION OF RADIOACTIVE COUPLED PRODUCTS ACCORDING TO THEINVENTION BY PRE-LABELLING THE LATEX-SUPPORT WITH ³ H OR ¹⁴ CETHANOLAMINE

Beads of "Estapor" were rendered radioactive by covalently binding ³ Hor ¹⁴ C ethanolamine to them. To 20 mg of the "Estapor" in suspension inBBS were added 0.05 ml of ¹⁴ C ethanolamine, 10μ Ci (1.6μ Mole) and 6.0mg of 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide in a total volumeof 4.0 ml. After rotary shaking for 2 hours at 20° C., 20μ moles of1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide were added. Theagitation was maintained for 2 hours then the contents of the tube weredialysed against BBS until free radioactivity had been eliminated.

The obtained product had a radioactivity of 2.2×10⁶ cpm per mg of"Estapor".

The fixation of antibodies was effected as described in Example 27. Theobtained product corresponded thus to a highly radioactive antibodypreparation in which the antibody has not sustained any chemicalmanipulation other than the fixation to latex of the carbohydratemoiety. The product can be used in all applications ofradioimmunological determination which use direct or indirect methods.The measurement of the reaction may be carried out by determining theradioactivity either of the agglutinated antigen-antibody complex, or ofthe unreacted beads, the separation being effected by centrifugation orfiltration.

These reagents (radioactive) allow the quantitative measurement ofantigens on the surface of cells to be done by counting theradioactivity adhering to the slides. Thus, it is possible to quantifythe data previously obtained when the beads were visualised under thescanning electron microscope.

In so far as it concerns the products derived from the coupling ofglyco-proteins and more particularly antibodies to supports comprisingat least one reactive --NH₂ through the intermediary of a carbohydrateresidue, which have been described in a non-limiting manner in Examples27 and 28, there is available thanks to the present invention, a rangeof beads containing antibodies which are specific for different antigensof interest to medical pathology and which allow rapid and easy serumdiagnosis of a wide range of illnesses. The use of beads containingantibody antigammaglobulins again widens the scope of their use inintroducing the indirect procedure. Quantification is easy and preciseeither by the direct dilution method or by the competitive method.

The procedure can be applied to all dosages (hormones, medicaments etc.)based on an immunological reaction. The use of radioactive beads rendersthe process as sensitive as all the other radioimmunological methodswhile diminishing at the same time the risk of modifying the antibodiesor antigens in the course of their iodination.

The described method presents, compared to currently utilised procedures(radioimmunological dosage, fluorescence, free radical estimation,immunoenzymological dosage, etc.), the following advantages: ease of usewithout necessitating particular equipment; sample manner of use thus nolong training periods are required; good sensitivity; stability ofreagents which can be kept for many months; and very low price.

EXAMPLE 31 PREPARATION OF LABELLED COUPLED PRODUCTS BY ISOTOPICLABELLING OF ANTIBODIES I.--LABELLING WITH TRITIUM

To 1.5 mg of goat antipolyamine gammaglobulins in 2 ml. of 0.14 M NaCl,0.02 M phosphatic buffer at pH 6 were added 0.65 ml of 0.06 M NaIO₄ insolution in a phosphate buffer of pH 6 (final concentration of NaIO₄ :0.015 M).

The mixture was left at 4° C. and protected from light for 3 hours andthen dialysed against a 0.14 M NaCl, 0.02 M phosphate buffer of pH 6 for1 hour with three changes, to eliminate excess NaIO₄ (verified with theaid of starch-iodide paper).

At this pH, the risk of forming Schiff's bases between the reactive--CHO aldehyde groups thus formed and the --NH₂ groups, either of lysineor of terminal --NH₂ groups of proteins, is reduced.

The contents of the dialysis sack was then transferred to a tube.

An excess of crystalline NaB³ H₄ (100 mCi/mMol obtained from New EnglandNuclear) was then added, followed by 1 ml of 0.1 M berate-HCl buffer ofpH 8.8 (this change of pH favours the reduction of --CHO groups to CH₂OH groups). After a contact time of 16 hours at 4° C., under a wellventilated hood, the contents of the tube were transferred to a dialysisbag and dial sed until the dialysis liquid was free of allradioactivity. One thus obtained a labelling of the antipolyamineantibodies of 670 cpm per μg of proteins.

It is also possible to increase the specific activity of the antibodiesin the following fashion:--to 1.5 mg of oxidised gammaglobulins wereadded 20μ Ci of ³ H ethanolamine (3.8 Ci/mMol) at a pH of 8.8; theSchiff's base thus formed were then stabilised by reduction with NaB³ H₄(5 mCi) for 18 hours at 4° C. the excess of NaB³ H₄ was eliminated bydialysis. This allowed one to obtain anti-polyamine gammaglobulinshaving a specific activity of 1807 cpm/μg.

¹⁴ C labelled antibodies can be obtained by a similar procedure butusing ¹⁴ C ethanolamine instend of ³ H ethanolamine and carrying out thereduction with NaB³ H₄.

II--Coupling of a solid insoluble support carrying side chains bearingat least one reactive --NH₂ to the --CHO groups of oxidisedgammaglobulins and stabilisation and lebelling of the Schiff's base soformed, by reduction with NaB³ H₄. The techniques used were thosedescribed in Examples 27 and 29 above.

As a result reagents were obtained which are of very long stability,which did not emit gamma rays (contrary to known procedures which relyon labelling by iodination of tyrosine groups in the protein chain,using radioactive iodine) and in which the biological activity isunaltered.

V.--EXAMPLES OF IMMUNO-REAGENTS EXAMPLE 32 PREPARATION OF A DIAGNOSTICREAGENT FOR MEASLES CONSTITUTED BY A PRODUCT DERIVED FROM THE REACTIONOF A SUBSTITUTED SUPPORT WITH OXIDISED MEASLES VIRUS I.--PREPARATION OFSUBSTITUTED COLOURED SUPPORT

(a) The attachment of a side chain carrying terminal --NH₂ groupsconstituted by a diamine is realised according to the proceduredescribed in Example 1 above.

(b) The fixation onto "ESTAPOR-NH₂ " obtained in the preceding step, ofan amino acid having an --SH group is effected using the proceduresdescribes in Example 16 above.

II.--PREPARATION OF OXIDISED MEASLES VIRUS

The oxidation of this virus is performed as described in the foregoingExample 20.

III.--COUPLING OF MEASLES VIRUS ON "ESTAPOR-SH"

To 20 mg of "ESTAPOR-SH" obtained in step Ib above in suspension in a0.1 M phosphate buffer of pH 6.0 were added 10 mg of oxidised measlesvirus as described in II above.

The mixture was stirred at 4° C. for 18 hours. It was then centrifugedat 6000 g for 60 minutes. The supernatant was recovered and the residuewashed twice with a phosphate buffer of pH 6.0.

The amount of protein remaining in the supernatant allowed one todetermine that 130 μg of viral protein had been fixed per mg of"ESTAPOR-SH"

EXAMPLE 33 PREPARATION OF A DIAGNOSTIC REAGENT FOR CANINE DISTEMPERVIRUS DISEASE CONSTITUTED BY THE PRODUCT OF THE REACTION BETWEEN ASUBSTITUTED SUPPORT WITH OXIDISED CANINE DISTEMPER VIRUS

The preparation of this diagnostic reagent was effected by the procedurecorresponding to that described in Example 32 I-II-III.

This reagent was tested under the conditions described in Example for"ESTAPOR" measles no agglutination was obtained in the presence of arabbit serum anti Canine distemper virus.

EXAMPLE 34 PREPARATION OF A DIAGNOSTIC REAGENT SPHERES OFLATEX-INFLUENZA VIRUS

The technique used for the preparation of this reagent was thatdescribed in Example 32 above, the measles virus being replaced withinfluenza virus.

EXAMPLE 35 PREPARATION OF A DIAGNOSTIC REAGENT SPHERES OF LATEX-GERMANMEASLES VIRUS

The same technique was used to prepare this reagent as that described inExample 32 for the preparation of diagnostic reagent for measles, butreplacing the measles virus with German measles virus.

EXAMPLE 36 PREPARATION OF A DIAGNOSTIC REAGENT SPHERES OFLATEX-CYTOMEGALIC VIRUS

To prepare this reagent the same technique was used as that described inExample 32 but replacing the measles virus but with cytomegalic virus.

EXAMPLE 37 PREPARATION OF "ESTAPOR" ANTIBODY DIAGNOSTIC REAGENTI.--PREPARATION OF THE SUBSTITUTED SUPPORT

The fixation on latex beads of a side chain having terminal --NH₂ groupsand the fixation on this chain of an amino acid having --SH groups wereeffected as described in Example 32.

II.--THE CHEMICAL OXIDATION OF THE GAMMAGLOBULIN IS PERFORMED ASDESCRIBED IN EXAMPLE 21 III.--COUPLING OF OXIDISED GAMMAGLOBULINS ON"ESTAPOR"-SH OBTAINED IN STEP I

This coupling was carried out according to the technique described inExample 29 above. By this technique, the coupling of gammaglobulinscontaining antipolyamine antibodies, gammaglobulins anti human fibrindegradation products (FPD) gammaglobulins anti human IgG, gammaglobulinsanti human IgM, gammaglobulins anti rabbit IgG and gammaglobulins- antiguinea pig IgG, have been achieved.

EXAMPLE 38 PREPARATION OF "ESTAPOR"-HUMAN CHORIONIC GONADOTROPHIN (HCG)I.--PREPARATION OF SUBSTITUTED ESTAPOR

The binding on latex beads of a side chain having terminal --NH₂ groupsand the attachment onto this chain of an amino acid having an --SH groupwere carried out as described in Example 16 above.

II.--PREPARATION OF OXIDISED HUMAN CHORIONIC GONADOTROPHIN

The oxidation was carried out in accordance with the technique describedfor gammaglobulins in Example 26 above.

III.--COUPLING OF ESTAPOR-SH TO CHORIONIC GONADOTROPHIN

This coupling was carried out in accordance with the technique describedin Example 29 above.

The effectiveness of the diagnostic reagents thus obtained isdemonstrated in the following Chapter VI.

VI.--EFFICIENCY OF THE IMMUNOREAGENTS OF THE INVENTION

The antigen antibody reaction is effected under conditions oftemperature, duration of contact etc., the most appropriate to eachparticular case. The positive reactions manifest is themselves byagglutination of the beads. Determining the results is effected byestimating the agglutination either simply by macroagglutination or bymicroagglutination on alveolar plates. It is possible to quantify thereaction either by direct nephelometric measurement or by centrifugationand visualization of the optical density of the remaining latex spheres,or by measurement of the radioactivity in the case of labelled latexespresent either on the filters or in the residue.

A/TESTS OF MACROAGGLUTINATION ON GLASS PLATES EXAMPLE 39 TEST OFAGGLUTINATING ACTIVITY OF "ESTAPOR--SH"-OXIDISED MEASLES VIRUS

The dilutions of antiserum were effected in the following buffer: 1%bovine serum albumin, 0.14 M NaCl: 0.01 M Tris-HCl at pH 8.2. Thereactive latex-measles virus reagent prepared according to the inventionwas also used in this test at a rate of 675 μg/ml.

The results obtained by macroagglutination on glass plates areillustrated in FIG. 4 in which the results obtained with the control(rabbit serum anti VSV) appear in the left hand side of the figure andthe results obtained with the rabbit serum anti measle, are found on theright hand side of the figure.

EXAMPLE 40 TEST OF AGGLUTINATING ACTIVITY OF "ESTAPOR-SH"-OXIDISEDCANINE DISTEMPER VIRUS

This reagent has been tested in the conditions reported in Example 39above.

Similar results have been obtained in the presence of rabbit serumanti-VSV as control, and of rabbit serum anti-Canine Distemper virus asthe sample tested.

EXAMPLE 41 TEST OF AGGLUTINATING ACTIVITY OF ESTAPOR-HCG BYMACROAGGLUTINATION ON A PLATE

Results: The reaction was positive with a rabbit anti-HCG serum dilutedto 1/160. At this dilution, inhibition of the reaction was obtained withthe urine of a pregnant woman.

EXAMPLE 42 TEST OF ACTIVITY OF REACTIVE SPHERES OFDOW-LATEX-ANTI-FIBRINOGEN BY MACROAGGLUTINATION ON PLATES

The spheres of Dow anti-FDP were suspended in the following buffer: 1%bovine serum albumin; 0.14 M NaCl; 0.1 M Tris-HCl at pH 8.2; 0.0015 Msodium azide at a concentration of 8 mg/ml. The reaction was positivewith human fibrinogen at 4 μg/ml and as shown in the attached FIG. 5 andalso with human serum diluted 1/10.

EXAMPLE 43 DETERMINATION OF THE POLYAMINE CONTENT IN URINE BYMACROAGGLUTINATION ON GLASS PLATES

The spheres of "Estapor"-polyamine prepared as described in Example 2above, are suspended in a buffer containing 1% bovine serum albumine,0.14 M NaCl, 0.1 M Tris, pH 8.3, 0.0015 M sodium azid, at aconcentration of 4 mg latex/ml.

A positive agglutination has been obtained with antipolyamine goat serumdiluted to 1/30 rd in the same buffer; the negative reaction has beenobtained with the same goat serum, before immunisation.

In those conditions, the positive reaction has been inhibited by theurin of pregnant woman.

B/ TESTS BY MICROAGGLUTINATION ON ALVEOLAR PLATES EXAMPLE 44 TEST OF THEMICROAGGLUTINATION ACTIVITY OF "ESTAPOR-SH"-OXIDISED MEASLES VIRUS

The results obtained using coloured Estapor prepared according toExample are shown in FIG. 3 of the attached drawings.

The antiserum dilutions were effected in the following buffer: 0.14 MNaCl, 0.01 M borate-HCl of pH 8.1.

A: Serum of rabbit J anti-measles prepared on Vero cells;

B: Serum of rabbit M anti-measles prepared on Vero cells;

C: Serum of rabbit B anti-VSV (VSV=Vesicular Stomatitis Virus) used ascontrol.

The dilution of serum was as follows:

    ______________________________________                                        Sample 1                                                                              1/10    Sample 5 1/160  Sample 9                                                                             1/2560                                 -- 2    1/20    -- 6     1/320  -- 10  1/5120                                 -- 3    1/40    -- 7     1/640  -- 11  1/10240                                -- 4    1/80    -- 8     1/1280                                               ______________________________________                                    

Sample 12 contained only buffer.

The reagent latex-measles virus according to the invention was used at arate of 675 μg/ml.

The reaction was positive for A₁ to A₆ and negative for A₇ to A₁₂.

The reaction was positive for B₁ to B₅ and negative for B₆ to B₁₂.

The reaction was negative for C₁ to C₁₂.

It may be seen from the test illustrated in FIG. 2 that the threecontrols A₁₂, B₁₂, C₁₂ are negative and that there is no non specificreaction with serum C.

The sensitivity of the reaction has been increased by submitting thelatex spheres-measles virus according to the invention to a preliminarytreatment with tritium X-100 at 1%: with the triton treated latexmeasles, the negative reaction with serum J does not start until A₁₀ andwith serum M until B₆. The reaction with serum C is totally negative.This sensitivity of reaction has been controlled by the tests withlatex-Vero (that is to say an extract of Vero cells used to prepare themeasles virus and coupled to latex under the conditions described in IIIof the present examples. There is not any reaction between the reactivelatex-Vero and the sera J, M and C which allows one to conclude that thereaction with the reactive latex-measles reagent prepared according tothe invention is specific and of a high sensitivity.

C/ QUANTIFICATION OF THE DIAGNOSTIC REACTION BY DIRECT NEPHELOMETRICREADING EXAMPLE 45 DIRECT MEASUREMENT BY NEPHELOMETRY FOR OXIDISEDGAMMAGLOBULINS COUPLED ONTO THE SUPPORTS OF THE INVENTION

To obtain a direct agglutination reaction of antipolyamine supports withantigens, one adds a fixed quantity of the reagent"Estapor"-antipolyamine of Example 37, namely 2 μg/ml in 0.1% of TritonX₁₀₀ -BBS buffer of pH 8.1 of known quantities (15 to 500 nanograms/mlof antigens). The mixture is left in contact for 45 hours. The lightdiffused at 90° by the mixture is measured. The results obtained areshown in FIG. 1 by Curve I for the normal rabbit gammaglobulins as oneantigen and by Curve II for the acid poly L glutamic spermine as theother, the quantities of antigen introduced being shown on the abscissaand the light diffused at 90° on the ordinates. The sensitivity and theprecision of measurement are of the order of nanograms.

EXAMPLE 46 TEST OF ACTIVITY OF A DIAGNOSTIC REAGENT "ESTAPOR" ANTICORPSBY NEPHELOMETRY

Dilutions of antigens and of latex antipolyamine antibodies wereeffected in "Borate BBS" buffer which had been filtered on a 0.22 μ"Millipore" filter (Borate Buffer Saline=0.01 M Na₂ B₄ O₇, 0.15 M NaCl,HCl at pH 8.1). After bringing into contact the reagents, the testsamples and the controls were incubated for one hour at 37° C. then atambient temperature.

Equilibrium was then attained at the 45th hour of incubation in the caseof 0.1 n mole of spermine coupled to of poly-L-glutamic acid (P.L.G.Spm) in the presence of 2 μg of latex-antipolyamine (latex coupled toantibodies from goats immunised against polyamines).

The specificity of this reaction has been shown by comparing theagglutination reactions obtained in the presence of P.L.G. Spm antigenwith latex-antipolyamine on one hand and latex coupled to rabbitglobulins as control on the other.

The application to the measurement of antigen necessitates thedetermination of the equivalent point: which is the maximum increase ofdiffused luminous intensity as a function of the ratio:latex-antibody/antigen.

This affinity at the equivalent point is a constant for a given batch oflatex-antibodies. It allows, knowing the concentration of latex-antibodyat the point of equivalence the deduction of the concentration ofunknown antigen present.

In practice, when confronted with a preparation of an antigen of unknownconcentration, it is preferable to make a range of concentrations oflatex-antibody.

On can prepare a control series and a series receiving a constantquantity of antigen.

I is the diffused luminous intensity for the controls. I' is thediffused luminous intensity for the test samples containing antigen.

I is a linear function of the concentration of latex-antibody in thetested region: from 62.5 ng of 8 μg/ml.

At each point of the range the ratio I'/I% translates the aggregationphenomenon of latex spheres in the presence of the correspondingantigen.

In the case of a preparation of latex coupled to globulins of goatsimmunised against human fibrinogen for 0.35 ngm of fibrinogen in thepresence of variable concentrations of latex-anti-fibrinogen, theequivalent point is attained for 200 mg of latex anti-fibrinogen, thatwhich corresponds to a ratio latex-antibody/antigen of 570. Thispreparation permits the estimation of a minimum quantity of 70 pg ofhuman fibrinogen with sufficient precision. The ratio at the equivalencepoint does not vary in a notable fashion in the course of keeping apreparation of latex-antibody for several months at 4° C. This isprobably due to the covalent bonding of the antibody to the latex and tothe elimination after the binding of antibodies which were simplyadopted.

D/ QUANTIFICATION OF THE DIAGNOSTIC REACTION BY READING OF THE OPTICALDENSITY EXAMPLE 47

To effect a direct agglutination reaction of "Estapor"-antipolyamineswith polyamines, one adds a fixed quantity of "Estapor"-antipolyamines(6 μg), prepared according to Example 27 above increasing quantities(from 31 to 2 000 nmoles) of antigens (spermine, spermidine, putrescinelinked to poly-L-glutamic acid and lysine HCl). The mixture is left for30 minutes at 25° C. then overnight at 4° C. After centrifugation at 3000 rpm for 15 minutes to sediment the "Estapor" beads and the antigenwhich is fixed to them, the optical density of the supernatant ismeasured at 230 nm. The results obtained are shown in FIG. 2, whichrepresents, in the order of reaction, curves I for spermine, II forspermidine, III for lysine, IV for putrescine and V for the blank, witha limit of detectability for spermine of 32 n moles, the optical densitybeing shown on the ordinates and the quantity in n moles of antigensadded on the abscissa.

E/QUANTIFICATION OF THE DIAGNOSTIC REACTION BY MEASUREMENT OF THERADIOACTIVITY OF LABELLED LATEXES EXAMPLE 48

The excellent sensitivity of reagents prepared according to the presentinvention has been demonstrated in the course of a comparative study ofagglutination carried out respectively with coupled products such asthose described by R. S. MOLDAY et al in the prior art (references citedabove) and with the radioactive coupled products obtained according toExample 30 above, which will be reported below.

COMPARATIVE STUDY OF AGGLUTINATION OF COUPLING PRODUCTS ACCORDING TOMOLDAY ET AL OF ONE PART AND ACCORDING TO THE INVENTION OF THE OTHERPART

I.--Some spherical carboxylated polystyrene beads of 0.30μ (referencePSI 68 of Rhone-Progil) rendered radioactive ¹⁴ C labelled ethanolaminewere substituted with hexamethylenediamine according to Example 1 andthen activated by reaction with 1.25% glutaraldehyde for 1 hour atambient temperature (20° C.), after which they were subjected toprolonged dialysis to eliminate the excess glutaraldehyde.

The substituted beads so obtained were estimated at 560 micrograms ofbeads per ml and their radioactivity was 95 000 cpm per ml.

520 micrograms of goat antibodies to rabbit antigammaglobulin were addedto it and left in contact therewith with agitation for 5 hours atambient temperature (20° C.).

II.--Some 0.30μ PSI 68 beads which had been prefiltered on a filter andlabelled with ¹⁴ C ethanolamine were substituted according to Example 10above with hexamethylenediamine and p-hydrazino-benzoic acid. 560micrograms of substituted beads were obtained in this manner per ml andtheir radioactivity evaluated at 87 000 cpm per ml.

To them was added 0.15 ml (i.e. 520 micrograms) of goat antibody torabbit antigammaglobulin which had been previously oxidised with sodiumperiodate according to the present invention.

In the two cases, the bead-antibody complexes were separated from freeantibody by centrifugation across a layer of 10% sucrose, the excesssucrose being eliminated by dialysis.

III.--Comparative study proper

Preparations I and II above were then treated as follows:

A/To 0.15 ml of each of the two preparations were added:

0.25 ml of 2% decomplemented calf serum

0.1 ml of rabbit serum at the following dilutions:

1/500

1/1 000

1/5 000

1/10 000

B/The composition of the controls was as follows:

0.15 ml of each of preparations I and II

0.25 ml of 2% decomplemented calf serum

0.1 ml of BBS buffer of pH 8.8.

The four tubes of the experimental series A and the tube of the controlseries B for each preparations I and II were incubated at 4° C. for 48hours, then 0.4 ml of each tube was filtered across a 0.80μ Milliporefilter presaturated with 2% decomplemented calf serum. The filters werewashed with 4×2 ml of 2% calf serum followed by 10 ml of BBS buffer andthen followed by 4×2 ml of BBS buffer.

The filters were counted in 10 ml of a mixture of Triton×100/toluene 1:2vol/vol.

The results obtained are set forth in the following Table.

                                      TABLE                                       __________________________________________________________________________          Maximum reactivity retained on the                                                             Progressive Diminution of the                          Preparation                                                                         filter, with sarum diluted to                                                                  radioactivity at dilutions                                                                     Equivalence Point                     __________________________________________________________________________    I     90 cpm               1/1000                                                                            1/5000                                                                            1/10000                                                                            1/500                                       dilution to 1/500                                                       II    113 cpm          1/500                                                                             1/1000       1/5000                                      dilution to 1/5000                                                      __________________________________________________________________________

It can be seen from the Table that: Compared to the fixation procedureaccording to the present invention, for which an equivalence point of1/5000 was obtained, the fixation of antibodies on latex particlesaccording to the technique of MOLDAY et al, for which an equivalencepoint of 1/500 was obtained implies that coupling by the lattertechnique brings about an important loss of sensitivity.

It results from the preceding that, whichever may be the ways ofcarrying out, of performing and of application adopted, there areobtained products resulting from the coupling of compounds containingcarbohydrate residues, through their --CHO group(s), with solid,insoluble supports bearing side chains carrying at least one reactive--NH₂ which reacts with the said --CHO group, to give rise to the saidcoupled product, which products can be used, notably as diagnosticreagents having a great stability and a very high sensitivity.

What I claim is:
 1. A process for agglutinating a biologically activeanalyte to an immunochemical reagent comprisingforming on an insolublesupport base a side chain having from three to ten carbon atoms and atleast one reactive --NH₂ group, by reacting a compound selected from thegroup consisting of diamines, polyamines, amino-acids, aliphatichydrazines bearing an acid group, and aromatic hydrazines bearing anacid group with an insoluble support base bearing at least one carboxylgroup or at least one amino group in the presence of a condensationagent to covalently bind said compound to said support base to form aside chain on said insoluble support base; oxidizing into a CHO group atleast one --CH₂ OH group of an organic compound having a carbohydrateresidue; attaching the organic compound to said support by reacting said--CHO group with at least one reactive --NH₂ on said side chain tochemically attach the organic compound on said support; and combiningsaid analyte and said support under conditions suitable foragglutination.
 2. A process according to claim 1, in which the compoundcovalently bound to the insoluble solid support is a diamine.
 3. Aprocess according to claim 2, in which the diamine is selected from thegroup consisting of hexamethylenediamine and diaminopropane.
 4. Theprocess of claim 1, further comprising coupling a nitrogen containingcompound having from 3 to 10 carbon atoms and at least one reactive--NH₂, to said side chain; said nitrogen containing compound selectedfrom the group consisting of aliphatic amines, aromatic amines,aliphatic hydrazines, aromatic hydrazines and amino acids, to form alonger side chain containing at least one reactive --NH₂.
 5. The processof claim 4, wherein said nitrogen containing compound is an amino acidcontaining both an NH₂ and a --SH group capable of forming ahetero-cyclic in the presence of the CHO group of the organic compoundto be attached.
 6. The process of claims 1, 4 or 5, wherein when saidreactive NH₂ is a terminal primary amine group, stabilizing theresulting Schiff base by chemical reduction.
 7. The process of claim 4,wherein said coupling is performed in the presence of a coupling agent.8. A process according to claims 1 or 7, wherein said condensation andsaid coupling agents are selected from the groups consisting ofglutaraldehyde, N-hydroxyl-succinimide and a totally or partiallywater-soluble carbodiimide of the general formula R--N═C═N--R in which Rrepresents an alkyl radical having 2 to 12 carbon atoms; a cycloalkylradical having 5 or 6 carbon atoms; a mono-aryl substituted lower alkylradical; a monoaryl radical; a lower alkyl radical substituted by amorpholinyl group, a lower alkyl radical substituted by a piperidylgroup, an ethylpiperidyl radical; a lower dialkylamino radical; a loweralkyl radical substituted by a pyridyle group, their acid addition saltswith acids and their quaternary ammonium salts, the two R radicals beingidentical or different.
 9. A process according to claim 7, in which thecoupling and condensation agents are added in two successive steps. 10.A process according to claim 4, in which the nitrogen-containingcompound is an amino acid selected from the group consisting ofβ-alanine, ε-amino-caproic acid, amino-acids having their carboxylgroups esterified by an N-hydroxylsuccinimide group, and amino-acidscarrying both --SH groups and --NH₂ groups capable of forming aheterocycle in the presence of the --CHO groups of the organic compoundto be bound onto said support.
 11. A process according to claim 4, inwhich the nitrogen-containing compound is a hydrazine selected from thegroup consisting of p-hydrazino-benzoic acid, and adipic dihydrazide.12. A process according to claims 1 or 4, in which the bound compound isa polyamine selected from the group consisting of spermidine,diaminopropylamine and spermine.
 13. A process according to claim 1, inwhich the bound compound is an amino-acid selected from the groupconsisting of β-alanine, ε-amino-caproic acid, amino-acids having theircarboxyl groups esterified by an N-hydroxylsuccinimide group, andamino-acids carrying both --SH groups and --NH₂ groups capable offorming a heterocycle in the presence of the --CHO group of the organiccompound to be bound onto said support.
 14. A process according toclaims 1 or 4, in which the amino compound attached by covalent bonds tosaid insoluble solid support is, a basic dye.
 15. A process according toclaims 1, or 4, in which the covalent binding of the side chains on thesupport is effected in a non-agglutinant buffer, containing no free--NH₂ groups and at a pH betweenn 6.0 and 8.8.
 16. A process accordingto claims 1, 4, 5, in which the covalent binding is followed by thedesorption of remaining non-coupled reagents.
 17. A process according toclaims 1, 4 or 5, in which the insoluble solid support is selected fromthe group consisting of latex spheres, agarose beads, dextran beads, andactivated glass beads.
 18. A process according to claims 1, 4 or 5, inwhich the insoluble solid support to which are bound the said sidechains, carries carboxyl groups, through the intermediary of which theside chains are bound on said support.
 19. A process according to claims1, 4 or 5, in which the solid insoluble support carries --NH₂ groupsthrough the intermediary of which the said side chains are bound on saidsupport.
 20. A process for chemically binding according to claims 1, 4or 5, wherein said oxidation of the --CH₂ OH group to a --CHO group, iseffected with sodium periodate.
 21. A process according to claims 1, 4or 5, in which the oxidation of the --CH₂ OH group to a --CHO group iseffected enzymically.
 22. A process according to claim 21, in which theenzyme used to carry out the oxidation of the --CH₂ OH group to a --CHOgroup enzymically is selected from the group consisting ofglucose-oxidase, fucose-oxidase and galactose-oxidase, where thelast-but-one residue of the carbohydrate chain is respectively glucose,fucose or galactose.
 23. A process according to claim 1, 4 or 5, inwhich the organic compound containing a carbohydrate residue issubjected, prior to the transformation of at least one of the --CH₂ OHgroups thereof to a --CHO group by oxidation, to a treatment withneuraminidase, when the terminal residue of the carbohydrate chain issialic acid, which treatment eliminates said sialic acid.
 24. A processaccording to any one of claims 1, 4 or 5, in which the binding of theorganic compound havig a carbohydrate residue is effected through theintermediary of said carbohydrate residue, by transforming two adjacentfunctional groups of said residue into --CHO functions, then causingthese --CHO groups and reacting said groups with a reactive --NH₂ of theside chains bound to the insoluble solid support.
 25. A processaccording to any one of claims 1, 4 or 5, in which the oxidationreaction of the carbohydrate residue of the organic compound to be boundto the support is effected at about pH
 6. 26. The process of claim 1further comprising using an oxidizing agent to aid in the oxidation. 27.A process according to claim 26, in which the excess of oxidising agentis eliminated at the end of the said oxidation reaction.
 28. Animmunochemical reagent for agglutinating a biologically active analyatecomprising a support base having a size which permits agglutination andhaving a side chain with 3 to 20 carbon atoms and at least one reactive--NH₂ covalently bound to said support base; said side chain havingattached thereto an organic compound being capable of agglutinating saidanalyte without reducing the biological activity of said analyte; saidside chain selected from the group consisting of aromatic amines,polyamines having at least three amine groups, amino-acids aliphatichydrazines bearing an acid group, aromatic hydrazines bearing an acidgroup, and a coupled compound formed by coupling a compound selectedfrom the group consisting of amines, polyamines, diacids, amino-acids,aliphatic hydrazines bearing an acid group and aromatic hydrazinesbearing an acid group, by the intermediary of the compound's reactive--NH₂, with a nitrogen-containing compound by carrying also at least onereactive --NH₂ and selected from the group consisting of aliphaticamines, aromatic amines, aliphatic hydrazines, aromatic hydrazines, andamino-acids; said support base selected from the group consisting oflatex spheres, agarose beads, dextran beads, and activated glass beads.29. A support according to claim 28, in which the side chains covalentlybound onto said support result from the coupling of a compound selectedfrom the group consisting of amines, polyamines, diacids, amino-acids,aliphatic and aromatic hydrazines bearing an acid group, by theintermediary of its reactive --NH₂, with a nitrogen-containing compoundcarrying at least one reactive --NH₂ and selected from the groupconsisting of aliphatic amines, aromatic amines, aliphatic hydrazines,aromatic hydrazines, and amino-acids.
 30. A support according to claim29, in which the insoluble solid support carries chains ofhexamethylenediamine (HMD) advantageously coupled through theintermediary of their reactive --NH₂ to adipic dihydrazide, orp-hydrazinobenzoic acid.
 31. A support according to claim 29, in whichthe insoluble solid support carries a side chain selected from the groupconsisting of spermidine, diamino-propylamine, spermine, and amino-acidshaving its carboxyl group esterified by a N-hydroxyl-succinimide group,and coupled through the intermediary of their reactive --NH₂, to adipicdihydrazide, p-hydrazino or benzoic acid.
 32. A support according toclaim 29, in which the insoluble solid support carries side chainscomprising an amino-acid selected from the group consisting of β-alanineand ε-amino-caproic acid coupled to diaminopropane, adipic dihydrazide,or p-hydrazino-benzoic acid.
 33. A support according to claim 29, inwhich said side chains are amino-acids carrying both --SH and --NH₂groups capable of forming a heterocycle in the presence of the --CHOgroup of the organic compound to be bound.
 34. A support according toclaim 33, in which the amino acid carrying both a --SH and a --NH₂group, bound to said support, is cysteine or one of its homdogues, whichforms in the presence of the --CHO group of the compound to be boundunto said support, a thiazolidine derivative.
 35. A colored supportaccording to claim 29, in which the side chains fixed to the supportresult from the coupling of an amine or polyamine with an acid function,and a basic dye selected from the group consisting of aliphatic amines,aromatic amines and hydrazines whereby the support is colored.
 36. Asupport according to claim 35, further comprising a non-agglutinatingbuffer to preserve said support.